While ex vivo T-cell depletion (TCD) is a highly effective methodology for prevention of lethal graft-vs.-host disease (GVHD), its clinical application is limited since the efficient removal of donor T-cells may compromise alloengraftment and an anti-leukemic effect. We have focused not on TCD but on the manipulation of T-cell function as a means of preventing GVHD. Advantages of functional manipulations include the possibility of inducing host antigen (Ag)/peptide-specific nonresponsiveness while preserving other donor cellular functions. For in vivo strategies, it is possible that residual host T-cells which can reject the donor graft may be affected similarly to the donor GVHD- causing cells. Since all T-cells would be affected, difficulties inherent to selective rather than pan T-cell subset manipulation can be avoided. We have chosen to interfere with the ability of T-cells to deliver the necessary intracellular signals for proliferation following encountering of host alloAgs. There are 2 main signalling pathways in T-cells. The classical pathway (signal 1) is triggered by ligation of the T-cell receptor (TCR) using anti-TCR mAbs or by peptide/MHC (major histocompatibility complex) molecules. Ligation of CD3epsilon chain is potent means of inducing TCR signals and T-cell proliferation if costimulatory molecules are also provided. To interfere with signal 1, we have infused an anti-CD3epsilon monoclonal antibody (mab) rendered non-mitogenic through removal of FcR. We have shown that anti- CD3epsilonF(ab')2 fragments which cannot bind to accessory cells (AC) (AC provide signal 2) are highly efficacious in preventing GVHD. Surviving recipients experience a pronounced non-destructive lymphoid inflammatory process at the site of tissue transplantation Ags. We will now ask: Will in vivo targeting or blockage of the CD28/CTLA4 signalling pathway be effective in preventing GVHD? Will Ag-specific non-responsiveness develop? If not, what cellular or soluble mediators are involved in overcoming anergy induction? For mice that develop infiltrating non- destructive cells, we will ask what is the nature and cause of the lymphoid tissue infiltrate in these recipients? What are the requirements for the induction of these cell populations? We will apply the principles of in vitro anergy (Ag-specific nonresponsiveness) induction to an in vivo GVHD system and ask: can (host) Ag-specific non- responsiveness be induced in vitro as a means of protecting murine recipients of allogeneic grafts from lethal GVHD? Is this GVHD protective effect restricted to certain GVHD types (eg. T-helper 1)? This proposal will provide important information on strategies to induce Ag-specific non-responsiveness in vivo.